The invention relates to short nucleic acid molecules specific to the the Bcl-XL mRNA that down-regulate the expression of the Bcl-XL protein by RNA interference and inhibit the growth of tumor cells in vivo, and to their use for cancer therapy.
Apoptosis is regulated in part by the Bcl-2 protein family, including pro-apoptotic Bax, Bcl-XS, Bak, Bad, Bid, Bik, Bim, and anti-apoptotic Bcl-2, Bcl-XL, Mcl-1, A1 and Bcl-W. The susceptibility of cells to apoptosis induced by various stimuli such as cytotoxic drugs, serum starvation and radiations appears to be determined, at least in part, by the subcellular localization and the relative ratio between pro- and anti-apoptotic proteins, which can heterodimerize and titrate one another's function (White, E., Genes Dev., 1996, 10, 1-15; Korsmeyer, S. J., Cancer Res., 1999, 59, 1693s-1700s; Cory, S. and Adams, J. M., Nat. Rev. Cancer, 2002, 2, 647-656).
High expression levels of anti-apoptotic members of Bcl-2 family have been found in many tumors, and up regulation of Bcl-2 and Bcl-XL has been shown to be a key element in malignancy and drug resistance (Reed, J. C., Semin. Hematol., 1997, 34, 9-19; Konopleva et al., Br. J. Haematol., 2002, 118, 521-534; Lebedeva et al., Cancer Res., 2000, 60, 6052-6060; Liu et al., Gynecol. Oncol., 1998, 70, 398-403; Simonian et al., Blood, 1997, 90, 1208-1206). In particular, Bcl-2 and/or Bcl-XL are frequently overexpressed in ovarian, nasopharyngeal, breast, prostate and colon carcinoma, glioma, mesothelioma, and melanoma (He et al., Chin. J. Cancer, 2003, 22, 11-15; Min et al, Chin. J. Oncol., 2004, 26, 14-16; Krishna et al., J. Neurosurg., 1995, 83, 1017-1022; Kajewski et al., Am. J. Pathol., 1997, 150, 805-814; Cao et al., Am. J. Respir. Cell. Biol., 2001, 25, 562-568; Soini et al., Clin. Cancer Res., 1999, 5, 3508-3515; Kojima et al., J. Biol. Chem., 1998, 273, 16647-16650; Olopade et al., Cancer J. Sci. Am., 1997, 3, 230-237; Zapata et al, Breast Cancer Res. Treat., 1998, 47, 129-140; Simonian et al., Blood, 1997, 90, 1208-1216; Leiter et al., Arch. Dermatol. Res., 2000, 292, 225-232; Nicholson et al., Nature, 2000, 407, 810-816; Krajewska et al., Cancer Res., 1996, 56, 2422-2427; Maurer et al., Dig. Dis. Sci., 1998, 43, 2641-2648; Mercatante et al., J. Biol. Chem., 2002, 277, 49374-49382; Ferrandina et al., Cancer Lett., 2000, 155, 19-27; Liu et al., Gynecol. Oncol., 1998, 70, 498-503; Marone et al, Clin. Cancer Res., 1998, 4, 517-524; French Patent Application FR 0108864). However, Bcl-XL is generally considered more efficient than Bcl-2 to suppress apoptosis induced by cytotoxic drugs and radiations (Simonian et al, precited; Gottschalk et al., P.N.A.S., 1994, 91, 7350-7354). Therefore Bcl-XL represents a good target for cancer therapy, especially for cancers resistant to conventional anticancer agents.
Transcription of the BCL2L1 gene (BCL2-like 1, BCL2L, BCLX, Bcl-X, bcl-x, or BCL-X gene) produces alternatively spliced variants encoding three isoforms: the longer anti-apoptotic isoforn (Bcl-XL), the shorter pro-apoptotic isoform (Bcl-XS) and a Bcl-X (beta isoform).
Bcl-XL is a 233 amino acids protein encoded by the longer transcript variant (transcript variant 1) comprising Exon 1, Exon 2 and Exon 3 of the BCL2L1 gene: the human Bcl-XL mRNA and protein correspond to NCBI accession numbers NP—612815 (SEQ ID NO: 3) and NM—138578 (SEQ ID NO: 2), respectively.
Bcl-XS is a 170 amino acids protein having the N-terminal sequence (positions 1 to 125) and the C-terminal sequence (positions 189 to 233) of the Bcl-XL protein but lacking a 63 amino acids sequence from positions 126 to 188 of the Bcl-XL protein. Bcl-XS is encoded by the shorter transcript variant (transcript variant 2) comprising Exon 1, the 5′ sequence of Exon 2 and Exon 3. Bcl-XS mRNA lacks the 189 nucleotides sequence located at the 3′ end of Exon 2, that is specific to the Bcl-XL transcript. Also, the corresponding 63 amino acids sequence of the Bcl-XL protein, which is missing in Bcl-XS, is specific to Bcl-XL protein. The human Bcl-XS mRNA and protein correspond to NCBI accession numbers NP—001182 and NM—001191, respectively.
Bcl-X (beta) is a 227 amino acids protein having the N-terminal sequence of Bcl-XL (positions 1 to 188 of Bcl-XL protein, encoded by Exon 1 and Exon 2 of the BCL2 gene), and a C-terminal sequence of 39 amino acids, encoded by Intron 2 of the BCL2 gene.
The Bcl-XL is located at the outer mitochondrial membrane and appears to regulate cell death by blocking the releases of the caspase activator and cytochrome c, from the mitochondrial membrane (Desagher S. and Martinou, J. C., Trends Cell. Biol., 2000, 10, 369-377; Hengartner, M. O., Nature, 2000, 407, 770-776; Kroemer, G., Nat. Med., 1997, 3, 614-620 ; Kroemer et al., Immunol. Today, 1997, 18, 44-51; Marchetti et al., J. Exp. Med., 1996, 184, 1155-1160; Minn et al., Nature, 1997, 385, 353-357; Susin et al., J. Exp. Med., 1996, 184, 1331-1341).
Down-regulation of Bcl-XL expression by antisense oligonucleotides was shown to induce apoptosis and to potentiate the cytotoxic effect of chemotherapy on cancer cells (US Patent Application US 2003/0191300; U.S. Pat. Nos. 5,776,905 and 6,143,291; International PCT Applications WO 00/01393, WO 00/66724; Lebedeva et al., precited ; Yang et al., The J. Biochem., 2003, 278, 25872-25878; Sonnemann et al., Cncer Letters, 2004, 209, 177-185; Sonnemann et al., Int. J. Oncol., 2004, 25, 1171-1181; Ozvaran et al., Mol. Cancer Therapeutics, 2004, 3, 545-550; Smythe et al., The J. Thoracic and Cardiovascular Surgery, 2002, 123, 1191-1198; Simoes et al., Int. J. Cancer, 2000, 87, 582-590, Hopkins-Donaldson et al., Int. J. Cancer, 2003, 106, 160-166; Heeres et al., Int. J. Cancer, 2002, 99, 29-34; Taylor et al., Oncogene, 1999, 18, 4495-4504; Wacheck et al., British Journal of Cancer, 2003, 89, 1352-1357; Taylor et al., Nature Biotechnology, 1999, 17, 1097-1100; Mercatante et al., precited; Frankel et al., Cancer Research, 2001, 61, 4837-4841; Roy et al., Oncogene, 2000, 19, 141-150). However, antisense oligonucleotide technology faces many problems including low absorption rates, non-specific inhibition effects, large effective dosage and toxicity.
The successful use of small interfering RNAs (siRNAs) technology for inhibiting the expression of a specific target holds great promise for the development of new treatment for cancer.
RNAi interference is the process where the introduction of double-stranded RNA into a cell inhibits gene expression in a sequence dependent fashion (reviewed in Shuey et al., Drug Discovery Today, 2002, 7, 1040-1046). RNAi has been observed in a number of organisms such as mammalian, Drosophila, nematodes, fungi and plants and is believed to be involved in anti-viral defense, modulation of transposon-activity and regulation of gene expression. RNAi is usually described as a post-transcriptional gene-silencing mechanism in which dsRNA triggers degradation of homologous messenger RNA in the cytoplasm. Target recognition is highly sequence specific since one or two base pair mismatches between the siRNA and the target gene will greatly reduce silencing effect. The mediators of RNA interference are 21-and 23-nucleotide small interfering RNAs (siRNA). In a second step, siRNAs bind to a ribonuclease complex called RNA-induced silencing complex (RISC) that guides the small dsRNA to its homologous mRNA target. Consequently, RISC cuts the mRNA approximately in the middle of the region paired with the antisens diRNA, after which the mRNA is further degraded.
Administration of siRNA to mice was shown to inhibit efficiently the expression of a target located in various organs (liver, brain, eyes, lung, kidney) as well as in xenografts (Braasch et al., Biochemistry, 2003, 42, 7967-7975; Duxbury et al., Biochem. Biophys., Res. Comm., 2003, 311, 786-792; Giladi et al., Mol. Ther., 2003, 8, 769-776; Lewis et al., Nat. Genet., 2002, 32, 107-108; Makimura et al, BMC Neurosci., 2002, 3, 18-; Reich et al., Mol. Vis., 2003, 9, 210-216; Song et al., Nat. Med., 2003, 9, 347-351; Zender et a., P.N.A.S., 2003, 100, 7797-7802.
Compared with antisense technology, RNAi is an efficient gene-specific technology, and has advantages of long-term stability, reversibility, and simple procedures.
RNAis targeting Bcl-XL have been used, in vitro to study the role of Bcl-XL in cell survival and resistance to chemotherapy. Down-regulation of Bcl-XL expression by RNAi was shown to induce apoptosis and to potentiate the cytotoxic effect of chemotherapy on cancer cells, in vitro (Taniai et al., Cancer Res., 2004, 64, 3517-3524; Zhang et al., Haematologica, 2004, 89, 1199-1206; Zender et al., Hepatology, 2005, 41, 280-288; Shimizu et al., Nature Cell Biology, 2004, 6, 1221-1228; Hon et al., J. Immunol., 2004, 173, 4425-4432; Dodier et al, Gynecologic Oncology, Epub, Sep. 26, 2005; He et al., Chinese J. Cancer, 2005, 24, 646-652; Liu et al., Acta Pharmacol., Sin., 2005, 26, 228-234; Lei et al., Acta Biochimica et Biophysica Sinica, 2005, 37, 555-560; Pizzi et al., Cell Death and Differenciation, 2005, 12, 761-772; Rorhbach et aL., J. Mol. Cell. Cardio., 2005, 38, 485-493; Tran et al., The J. Biol., Chem., 2005, 280, 3483-3492; Zhu et al., Cancer Biology and Therapy, 2005, 4, 393-397; Zhu et al., Molecular Cancer Therapeutics, 2005, 4, 451-456; US Patent Application 2005/0176025).
However, few RNAis are specific to Bcl-XL (Zender et al.; Hon et al.; Dodier et al.; He et al., Liu et al., Tran et al., Zhu et al., Cancer Biology and Therapy, precited). Furthermore, some of the Bcl-XL-specific siRNAis have been tested only in combination with non-specific siRNAis (Dodier et al. precited) and none of the Bcl-XL-specific siRNAis was proven to have an antitumoral effect in vivo.